The contribution of L3T4+ T cells to lymphoproliferation and autoantibody production in MRL-lpr/lpr mice.

The current study examines the role of the L3T4 T cell subset in the development of lupus-like autoimmunity and lymphoproliferation in lpr-bearing mice. Chronic treatment of MRL-lpr/lpr mice with anti-L3T4 antibody beginning at 4 wk old was found to markedly decrease the production of IgG anti-DNA and antihistone antibodies, while having no effect on IgM autoantibodies. A dramatic reduction in splenomegaly and lymphadenopathy was also observed coincident with a decrease in the percentage and total number of Thy-1+, B220+ cells. Together, the data suggest an important role for L3T4+ T cells in the pathogenesis of disease in lpr mice and provide further evidence that a requirement for the L3T4 subset may be a common feature of murine autoimmunity.

Aberrant production of leukotriene C4 by macrophages from autoimmune-prone mice.

Eicosanoids have been implicated in the pathogenesis of autoimmune diseases. In this study, peritoneal macrophages from autoimmune-prone mice were examined for their capacity to produce proinflammatory 5-lipoxygenase metabolites. The results indicate that enhanced production of leukotriene C4 is a common feature of murine autoimmunity and suggest further that aberrations in 5-lipoxygenase activity may play a role in the development of lupus.

Incorporation, distribution, and turnover of arachidonic acid within membrane phospholipids of B220+ T cells from autoimmune-prone MRL-lpr/lpr mice.

The metabolism of AA-containing phosphoglycerides within T cell membranes leads to the generation of second messengers that appear to play a crucial role in transmembrane signal transduction. To test the hypothesis that aberrations in the movement of arachidonoyl-phospholipids are associated with and may potentially contribute to abnormal T cell function, the incorporation, distribution, and turnover of AA within the membrane glycerolipids of cells that are known to exhibit immunoregulatory disturbances was examined. Thy-1+, Ly-1+, L3T4-, Lyt-2-, B220+ T cells from autoimmune MRL-lpr/lpr mice were used as the cellular model. In contrast to control lymph node T cells, which preferentially incorporate labeled AA into phosphatidylcholine (PC), B220+ T cells displayed a predilection for distributing [3H]arachidonate into phosphatidylinositol (PI). The arachidonoyl-phospholipid pools were normal in B220+ T cells. The constitutive turnover of [3H]arachidonoyl-PI was significantly enhanced and that of [3H]arachidonate-PC substantially reduced in B220+ T cell compared with control cells. Using membrane homogenates B220+ T cells demonstrated a functional increase in the levels of lyso-PI. Intact B220+ T cells prelabeled with [3H]myoinositol and cultured in the absence of stimulation with exogenous antigens or mitogens, exhibited increased production of lyso-PI. The data indicate that the preferential formation of [3H]arachidonoyl-PI in B220+ T cells is the result of greatly increased, constitutive PI turnover that appears to be due to a membrane phospholipase A2 activity. It remains possible that disturbances in the movement of arachidonate within phospholipids of B220+ T cells play a role in the expression of aberrant immunological activity.

Long chain fatty acid utilization of T-cells from autoimmune MRL-lpr/lpr mice.

To test the hypothesis that T-cells which exhibit abnormal immunological behavior manifest derangements in the de novo synthesis of phospholipids, the utilization of [3H]palmitic acid in B220+ T-cells from autoimmune MRL-lpr/lpr mice was investigated. The rate of incorporation of [3H]palmitic acid into membrane phospholipids was markedly increased in intact B220+ T-cells compared to that in T-cells from immunologically normal mice. The activities of two key enzymes involved in the de novo synthesis of palmitoyl-phospholipids, acyl-coenzyme (CoA) ligase and acyl-CoA; sn-glycerol-3-phosphate acyl transferase, were significantly higher in homogenates from B220+ T-cell membranes compared with those in controls. Despite these findings, the molar concentration of individual palmitoyl glycerolipids was equivalent in the membranes of B220+ T-cells and control lymph node T-cells. The results indicate that T-cells from lupus mice exhibit complex defects in the biosynthesis and turnover of membrane phospholipids and suggest the possibility that these aberrations contribute to T-cell dysfunction in autoimmune diseases.

Aberrant cytokine gene expression in the hippocampus in murine systemic lupus erythematosus.

Cytokines are important mediators of immune regulation and have been implicated in the pathogenesis of the neurological disturbances, which occur in up to sixty percent of patients with systemic lupus erythematosus (SLE). SLE is an autoimmune disease characterized by the presence of autoantibodies against nuclear antigens, including native DNA. Cytokines are thought to drive autoantibody production in lupus. Certain of the derangements in memory and learning described in human and experimental SLE map to the hippocampus. The current study examines the expression of cytokine genes in the hippocampus in lupus, using MRL-lpr/lpr mice as the experimental model. These mice spontaneously develop a SLE-like illness accompanied by disturbances in spatial learning. Our results suggest a potential role for proinflammatory cytokines in the cognitive aberrations observed in lupus.

Regional influences on the physical properties of T cell membranes.

Differences in the composition of membrane lipids are well documented between cells from distinct tissues. These differences may be manifested by changes in the motional freedom or fluidity of lipid molecules within plasma membranes and may predispose to alterations in cellular function. Regional influences on immune function have been implied by the finding that thymic-derived cells from murine spleen and lymph nodes are differentially responsive to antigen priming. The possibility that microenvironment also shapes the physical properties of T lymphocyte membranes has not been explored and is the focus of this study. Using mice as the experimental model, differences were found in fluidity and in the resting level of intracellular free-ionized Ca2+ between splenic and lymph node T cells from immunologically normal mice and from autoimmune-prone MRL-lpr/lpr mice. The results indicate that T cells are more heterogeneous than previously recognized and suggest a potential role for microenvironment in determining immune responsiveness.

Constitutive turnover of inositol-containing phospholipids in B220+ T cells from autoimmune-prone MRL-lpr/lpr mice.

B220+ T cells from mice that are homozygous for the lpr gene exhibit profound defects in their capacity to produce and respond to IL-2 and provide a cellular model for investigating the basic requirements for effective transmembrane signal transduction in immunologically normal T cells. A correlation between defective lectin-stimulated proliferation and deficient hydrolysis of inositol-containing phospholipids (PI) has recently been demonstrated in B220+ T cells. The finding has been postulated to explain abnormal expression of protein kinase C (PKC) activity in these cells. In a previous study, we found that the constitutive turnover of [3H]arachidonyl-PI was markedly increased in B220+ T cells from lpr-bearing MRL mice relative to that in controls. This observation suggested that an inability to metabolize PI and to generate second messengers putatively necessary for transmembrane signaling might not be responsible for aberrant PKC activity in B220+ T cells. To clarify this issue, the constitutive turnover of phosphoinositides in B220+ T cells from autoimmune-prone MRL-lpr/lpr mice was investigated. We found that in the absence of stimulation with exogenous Ag, B220+ T cells exhibited greatly increased 1) incorporation of labeled myoinositol into PI, 2) production of inositol phosphates in cells prelabeled with [3H]myoinositol, and 3) formation of diacylglycerol in [3H]arachidonic acid-labeled cells. Increased spontaneous PI turnover in B220+ cells was associated with normal phosphatidyl inositol-4,5-biphosphate-phospholipase C activity in membrane homogenates, normal levels of membrane PI, and normal resting and mitogen-stimulated levels of intracellular free-ionized Ca2+. The results suggest that an incomplete form of the PI cycle, one unassociated with PKC activation, is constitutively expressed in B220+ T cells.

T cells from autoimmune "IL 2-defective" MRL-lpr/lpr mice continue to grow in vitro and produce IL 2 constitutively.

MRL-lpr/lpr mice and other autoimmune strains that bear the lpr gene develop a profound lymphadenopathy characterized by an expansion of a unique dull Lyt-1+2- T cell population. Because fresh splenic and lymph node T cells from such mice stimulated Con A in vitro are extremely defective in IL 2 production and proliferation, T cell lines derived from MRL-lpr/lpr spleens were established and maintained for several months, and were analyzed for their factor production to define their growth requirements. The results indicate that cultivation in vitro leads to constitutive production of IL 2 and the capacity to respond to growth factors, thereby facilitating the continuous proliferation of T cells bearing the dull Lyt-1+2- phenotype in vitro in the absence of exogenous antigen or mitogen. These studies indicate that MRL-lpr/lpr T cells have the ability to produce IL 2 and to respond to IL 2 with long-term proliferation. In addition, the impaired responsiveness to Con A of fresh MRL-lpr/lpr lymph node T cells was found to be quite transitory, because even short-term culture allowed MRL lpr/lpr T cells to respond normally.

The basis of autoimmunity in MRL-lpr/lpr mice: a role for self Ia-reactive T cells.

Murine models of systemic lupus eRythematosus (SLE) have significantly contributed to our understanding of human autoimmunity. One such strain, the MRL-lpr/lpr, spontaneously develops an autoimmune disease manifested clinically by arthritis, vasculitis, immune-complex glomerulonephritis and autoantibody production(1-3). In this article Yvonne Rosenberg and her colleagues suggest a theoretical basis for the development of autoimmunity in MRL-lpr/lpr mice.

The induction of tolerance and suppression in autoimmune MRL mice using hapten-modified self.

Disturbances in suppressor cell function have been considered important in the pathogenesis of systemic lupus erythematosus (SLE), a conclusion supported by studies with New Zealand mice. To determine whether other SLE mice display similar immunoregulatory defects, we investigated the susceptibility of autoimmune MRL mice to unresponsiveness induced by hapten-modified self (HMS). The response of splenocytes from MRL-lpr/lpr mice (lpr) was compared with those of sex- and age-matched, congenic MRL-+/+ mice (+/+), and H-2-identical (H-2k) CBA/J mice. Spleen cells (NSC) were cultured in vitro with hapten-modified syngeneic splenocytes (TNP-SC) and tested for responsiveness to TNP-LPS (for tolerance) or their ability to suppress the response of fresh cells. There was no difference in the susceptibility of lpr splenocytes from 3- and 10-mo-old mice to the induction of tolerance or suppression when compared with those from age-matched +/+ or CBA mice. To evaluate any quantitative defects in the responsiveness of lpr splenocytes to HMS, we modified the conditions under which suppressor activity was generated. Varying the ratio of NSC to TNP-SC from 10:1 to 2000:1, or changing the concentration of TNBS for haptenation from 10 mM to 0.5 mM per 10(8) spleen cells revealed no differences in the dose-response curves of lpr splenocytes for both tolerance and suppression when compared with those of the CBA. These results indicate that clinically affected MRL mice have intact suppressor cell activity in response to antigen-modified self and suggest a possible therapeutic role of this modality in inducing tolerance to self-antigens.

Idiopathic anasarca associated with oligoclonal gammopathy in systemic lupus erythematosus.

We describe a young woman with systemic lupus erythematosus with acute onset of severe idiopathic anasarca and the appearance of serum IgG kappa oligoclonal bands. We hypothesize that her anasarca was due to a defect in capillary permeability, perhaps related to the clonally restricted immunoglobulins detected in her serum.

Interleukin 2 is a proliferative signal for B cells from autoimmune mice.

T cells from murine lupus strains manifest complex defects in interleukin 2 (IL 2) production and receptor expression. The capacity of B cells from such mice to utilize IL 2 as a growth factor has not been previously reported and is examined herein. Anti-Thy-1.2 plus complement-treated spleen cells from 6-8-week-old autoimmune MRL-lpr/lpr mice and from age and sex-matched immunologically normal CBA/J mice were cultured with lipopolysaccharide (LPS) for 36 h and analyzed for the expression of IL 2 receptors using the monoclonal antibody 7D4. The percentage of B cells expressing IL 2 receptors was comparable in MRL-lpr/lpr and CBA/J mice. In contrast to those from CBA/J, BALB/c and (BALB/c X NZW)F1 mice, LPS-stimulated B cells from MRL-lpr/lpr and from (NZB X NZW)F1 mice were capable of proliferating in response to IL 2. Fractionation of MRL-lpr/lpr B cells using Percoll gradient density separation demonstrated that the IL 2-responsive population consisted predominantly of large cells. In addition, unfractionated B cells from MRL-lpr/lpr mice were found to be substantially more responsive to IL 2 than those from CBA/J and BALB/c mice following activation with anti-immunoglobulin plus LPS. The hyper-responsiveness to IL 2 may be a consequence of the state of activation of autoimmune B cells and is of potential importance in the pathogenesis of systemic lupus erythematosus.

The role of L3T4+ cells in the pathogenesis of lupus in lpr-bearing mice. I. Defects in the production of interleukins 2 and 3.

Mice which bear the lpr gene spontaneously develop autoimmune syndromes characterized by massive expansion of an unusual T cell subset which is phenotypically Thy-1+, L3T4-, Lyt-2-, B220+. The mutant T cells are refractory to stimulation with mitogenic lectins and, by implication, are thought to be solely responsible for the defects in lymphokine production manifested by lpr mice. The contribution of the remaining L3T4+ T cell subset to the latter derangements has not been previously examined and is the focus of this study. We found that abnormalities in concanavalin A-induced interleukin 2 and 3 production in the spleens of MRL-lpr/lpr and C57BL/6.lpr mice occurred in the presence of limited infiltration with B220+, L3T4- T cells. Mixing experiments indicated that B220+ T cells were not suppressive. Furthermore, lpr spleen cells enriched for L3T4+ cells and depleted of sIg+, B220+ and Lyt-2+ cells demonstrated reductions in lymphokine production which were comparable to those seen in unfractionated preparations. Spleen cells from C57BL/6.lpr mice, enriched for L3T4+ cells, were also markedly impaired in a mixed leukocyte reaction in response to stimulator cells from the class II major histocompatibility complex mutant bm12. The results indicate that the aberrations in lymphokine production and proliferation in the spleen cells of lpr mice involve not only B220+ T cells but also L3T4+ cells and suggest a potential role for the L3T4+ subset in the pathogenesis of lupus in lpr-bearing mice.

Cerebellar dysfunction is associated with overexpression of proinflammatory cytokine genes in lupus.

Systemic lupus erythematosus (SLE) is an autoimmune disease of unknown etiology accompanied by central nervous system involvement in up to 60% of patients. The current study chronicles the expression of cerebellar dysfunction in SLE using MRL-lpr/lpr mice as the experimental model. These mice spontaneously develop an illness that has immunological and clinical features of human lupus. We found that MRL-lpr/lpr mice manifest severe and progressive behavioral disturbances indicative of cerebellar dysfunction beginning at 11 weeks of age. Although the lpr gene is known to induce autoimmune features, immunologically normal mice rendered congenic for lpr failed to exhibit disturbances in cerebellar function. Because lupus is a cytokine-driven disease and overexpression of certain proinflammatory cytokines has been associated with neurodegeneration, the relationship between cerebellar dysfunction and cytokine gene expression was examined. Relative to immunologically normal CBA/J mice, the cerebellum of young (11-15 weeks of age) MRL-lpr/lpr mice contained high levels of interleukin (IL)-6 and interferon-gamma (IFNgamma) mRNA, which became even more pronounced in old (22-30 weeks of age) autoimmune mice. mRNA levels for the cytokines IL-1beta and IL-10 were elevated in the cerebellum of old, but not young, MRL-lpr/lpr mice relative to CBA/J. In contrast, the levels of cerebellar transcripts for IL-3 and tumor necrosis factor-alpha were comparable in autoimmune and normal mice, indicating that enhanced gene expression of IL-6, IFNgamma, IL-1beta, and IL-10 was selective. These results suggest a potential role for certain proinflammatory cytokines in the pathogenesis of cerebellar disturbances in SLE.

The cellular basis for immune interferon production in autoimmune MRL-Ipr/Ipr mice.

Disturbances in immune interferon (IFN gamma) activity have been implicated in the development of human systemic lupus erythematosus (SLE) and the spontaneous disease sustained by autoimmune-prone mice. We therefore investigated the cellular basis for IFN gamma production in MRL-Ipr/Ipr mice and examined the relationship between synthesis of interleukin 2 (IL 2) and IFN gamma. In vitro IL 2 and IFN gamma production in 3 to 6-mo-old, autoimmune MRL-Ipr/Ipr and MRL-+/+ mice was compared with that seen in age- and sex-matched, immunologically normal CBA/J mice. 5 X 10(6) spleen cells were pulsed with 5 micrograms of concanavalin A (Con A), and the cellfree supernatant was assayed for IL 2 and IFN gamma activity at various times up to 72 hr. We found that peak levels of IL 2 in MRL mice were less than 10% of those in the CBA/J. Yet, production of IFN gamma by cells from the autoimmune and normal strains was quite comparable. The addition of murine IL 2 to optimally Con A-stimulated cells from the MRL-Ipr/Ipr or normal mice did not affect the subsequent peak production of IFN gamma. Although the primary producers of IFN gamma in cultures of normal mice bear the Lyt-2+ phenotype, the Lyt-1+2- T-cell subset was found to be the principal source of IFN gamma in the aged MRL-Ipr/Ipr. These data suggest that Lyt-1+ cells from MRL-Ipr/Ipr mice may be differentially responsive to the signal delivered by the same mitogenic lectin with respect to lymphokine production and may indicate a distorted commitment of such cells toward production of IFN gamma and repression of IL 2 synthesis. The relationship between hypoproduction of IL 2, this usual source of IFN gamma, and the autoimmune disease sustained by MRL-Ipr/Ipr mice remains unclear.

Studies of congenic MRL-Ipr/Ipr.xid mice.

Highly inbred MRL-Ipr/Ipr.xid congenic mice were bred and compared with their + littermates. The xid-bearing congenics developed lymphadenopathy consisting of dull Ly-1+ T cells and impairment of cellular proliferation and IL 2 production in response to the T cell mitogen Con A. Thus, the lpr gene was fully expressed. The xid gene, however, was also expressed as indicated by the failure to respond to immunization with TNP-Ficoll and flow cytometric analysis of splenic B cells. The xid gene was associated with a marked reduction in IgM anti-ssDNA and anti-nDNA of both classes, and serum Ig-bound gp 70. Kidney disease was markedly retarded as was death from the autoimmune process. These studies suggest that the T cell lymphoproliferation and dysfunction characteristic of MRL-Ipr/Ipr mice is not sufficient to induce accelerated autoimmunity; xid is able to markedly slow the process. The xid gene interferes with the development of a B cell subset necessary for maximum autoantibody production, anti-gp 70 production, and the resultant immune complex renal and cardiac disease. The present finding of protection against accelerated autoimmunity in MRL-Ipr/Ipr mice by xid, coupled with previous demonstrations of protection against autoimmunity in other autoimmune mouse strains, suggests that a common approach to the therapy of systemic lupus may be possible.

Aberrant lymphocyte trafficking in murine systemic lupus erythematosus.

The patterns of migration of lymphoid cells from autoimmune-prone MRL-lpr/lpr, C57BL/6-lpr/lpr, MRL-+/+, and NZB mice were compared to those from sex and age-matched, normal CBA, C57BL/6, and BALB/C mice. Chromium-51-labelled spleen and lymph node cells from all autoimmune mice tested homed preferentially to the spleen relative to lymph node of the recipient strain. The data indicate that defects in lymphocyte trafficking are widespread in murine lupus and suggest a role for abnormal lymphocyte migration in the pathogenesis of this disease.

Studies of lymphoproliferation in MRL-lpr/lpr mice.

MRL-lpr/lpr mice develop massive lymphoproliferation and an associated autoimmune process that includes anti-DNA formation, vasculitis, and glomerulonephritis. We have investigated the lymphoproliferation of MRL-lpr/lpr mice and have found that multiple factors are operative. Although neonatal thymectomy markedly retards the syndrome, chronic injection of poly rI.rC could substitute for the thymus. The resulting cells had the phenotype characteristic of the abnormal MRL-lpr/lpr T cells, Thy-1+, dull Ly-1+, Lyt-2-, 6B2+, Ig-. Splenectomy at 2 wk of age markedly retarded the development of this syndrome; however, splenectomy at birth did not. Some protection was afforded by splenectomy at 5 wk. Thus, there appears to be a critical period in the life of an MRL-lpr/lpr mouse when the spleen contributes importantly to the lymphoproliferation. A most remarkable observation was that an MRL-lpr/lpr spleen graft under the kidney capsule could induce lymphadenopathy characteristic of lpr/lpr mice in MRL +/+ recipients. Cells within the graft had to be able to proliferate for the adenopathy to occur because irradiation of the spleen with 800 R just before grafting abrogated the lymphadenopathy-inducing potential. No adenopathy was induced by +/+ spleen grafts placed into +/+ mice. Although MRL-lpr/lpr males develop disease slightly more slowly than female littermates, the differences are small. Manipulations that retard disease, such as splenectomy at 2 wk or neonatal thymectomy, magnified the sex differences. Male MRL-lpr/lpr mice that were thymectomized and splenectomized and given polyclonal immune activators failed to develop either anti-DNA or lymphadenopathy, whereas their female littermates expressed both abnormalities. We conclude from these studies that multiple factors serve to modulate the magnitude of the lymphoproliferation and autoimmune syndrome of MRL-lpr/lpr mice.